PROJECT SUMMARY/ABSTRACT Age is the strongest risk factor for Alzheimer?s disease (AD). However, cell and animal models of AD fail to recapitulate human aging and fail to capture key aspects of disease pathology. Our long-term goal is to understand how aging contributes to AD pathogenesis. Therefore, we propose to develop an induced pluripotent stem cell (iPSC)-based model of AD that incorporates accelerated aging, with the objective of more robustly modeling AD onset and progression. Recent findings indicate that perturbations in Lamin A biology may contribute to AD. In preliminary studies, we have found a significant increase in LMNA, the gene that encodes the nuclear envelope protein Lamin A, and a significant decrease in ZMPSTE24, a prelamin A processing enzyme, in autopsy-confirmed AD brain tissue and in laser-dissected neurons from AD brains compared to age- matched controls. We predict that these changes in LMNA and ZMPSTE24 levels would cause an increase in farnesylated prelamin A, which has been shown to drive accelerated aging phenotypes, including acquisition of an abnormal nuclear lamina, and impairments in nucleo-cytoplasmic compartmentation, chromatin organization and gene expression similar to the accumulation of the LMNA isoform progerin. We hypothesize that forced expression of progerin accelerates age-associated changes and disease phenotypes in iPSC-cortical cells derived from AD patients. In our studies using R406W MAPT isogenic pairs, progerin over expression caused distorted nuclei, reduction in heterochromatin, increased DNA damage and a concomitant reduction in Lamin B1 in both neurons and astrocytes. In addition, R406W mutant derived cultures showed higher rate of cell death and loss of synapses following progerin over expression. Synapse loss is a well-recognized feature of AD and related dementias, and given the exacerbation of this with progerin treatment, we are focused on this important observation. In order to study synapses effectively in the collection of patient-derived iPSC lines, we needed to establish a cortical differentiation protocol in which synaptic markers appear robustly and earlier compared to other protocols. We now propose to perform an in-depth transcriptomic and functional analysis of cortical cultures generated with this new protocol. Using this improved in vitro synaptogenesis iPSC-cortical model, we will assess and quantify the effects of forced progerin expression on cell phenotypes related to AD pathology, including A? and tau secretion, aggregation and turnover, in iPSC-2D cortical cells and in 3D cortical organoids. We will also assess the impact of introducing microglia into 3D organoids and study microglia mediated synapse loss. This supplemental work to characterize and utilize the improved cortical synaptogenesis protocol will significantly enhance our ability to model AD using progerin-induced accelerated aging. The findings will open new avenues for investigating the contribution of aging to disease mechanisms underlying AD pathology and will advance the development of in vitro 2D and 3D models of AD to aid potential therapeutic strategies.